Repression and activation of promoter-bound RNA polymerase activity by gal repressor 1 1Edited by R. Ebright

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Repression of transcription initiation from the two gal promoters, P1 and P2, requires binding of GalR protein to two flanking operators, O E and O I , binding of HU to a site, hbs, located between the two operators, and supercoiled DNA template. Previous experiments suggested that repression involves the interaction of two DNA-bound GalR proteins, which generates a 113-bp DNA loop encompassing the promoter region. Interaction between two DNA-bound proteins would be allowed if the binding sites on DNA are properly aligned. To test the idea that the observed repression of gal transcription in vitro is mediated by DNA looping, we investigated the effect of changing the relative angular orientation of O E and O I in the DNA helix. We found that repression is a periodic function of the distance between the two operator sites. Since repression recurred commensurate with DNA helical repeat, we conclude that the observed in vitro repression is mediated by DNA looping and the in vitro conditions reflect the in vivo situation.DNA looping appears to be a general phenomenon in DNA transaction reactions e.g. replication, transcription regulation, site-specific recombination, DNA condensation, etc. In gene regulation, DNA looping is frequently involved in both transcription repression and activation. Looping occurs when two identical or different proteins bound to spatially separated specific sites on DNA interact directly or through a mediator or when a bidentate protein simultaneously binds to distal DNA sites. Depending on the strength of the above interactions, DNA looping may or may not require the aid of a DNA bending protein (frequently referred to as an architectural protein), which facilitates loop formation by binding to a locus between the two distal DNA sites. In the loop, the two binding sites on DNA may be oriented in parallel or in antiparallel orientation (1-3).The feasibility and stability of such a DNA-multiprotein complex has been studied in several systems, including the gal, lac, and ara operons and bacteriophage of Escherichia coli. The complex formation depends on a variety of conditions: the proper angular orientation of the protein binding sites on DNA (4 -8), the size of the loop (9), and the superhelicity of the DNA (10 -12). Because shorter DNA resists torsional change (13), the proper angular orientation of the two binding sites is more important with relatively shorter loop size than with longer loop size. It has been suggested that a loop size of less than 150 bp strictly depends upon proper phasing of the protein binding sites, less so for 200 bp, and not at all for 400 bp and up. The precise size of the helix repeat also depends to a certain extent on the DNA sequence of the loop (9). Here we report the results of the effect of changing the relative angular orientation of the binding sites, which were separated by less than 150 bp in DNA, on transcription repression in the gal operon of E. coli (14). Previous results suggested that repression involves the formation of a DNA loop by the intera...

Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 96%

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In Vitro Repression of the gal Promoters by GalR and HU Depends on the Proper Helical Phasing of the Two Operators

Lewis

Adhya

2002

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“…There is to our knowledge, however, no evidence that, in any of the systems for which regulation by loop formation has been described, trapping of RNAP causes repression. A mechanistic parallel exists with several other transcription factors, which have been shown to cause repression of transcription initiation by stabilization of either the closed complex (GalR at P1 in the gal operon (45,46)) or the open complex (p4 at the 29 A2C promoter (47)). The observed stabilization in those cases, however, is due to specific contacts between the repressor and the ␣-CTD of RNA polymerase.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for H-NS-mediated Trapping of RNA Polymerase in the Open Initiation Complex at the rrnB P1

Dame

Wyman

Wurm

et al. 2002

164

138

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in both transcription regulation and DNA compaction. Each of these processes involves H-NS-mediated bridge formation between adjacent DNA helices. With respect to transcription regulation, preferential binding sites in the promoter regions of different genes have been reported, and generally these regions are curved. Often H-NS binding sites overlap with promoter core regions or with binding sites of other regulatory factors. Not in all cases, however, transcriptional repression is the result of preferential binding by H-NS to promoter regions leading to occlusion of the RNA polymerase. In the case of the rrnB P1, H-NS actually stimulates open complex formation by forming a ternary RNAP⅐H-NS⅐DNA complex, while simultaneously stabilizing it to such an extent that promoter clearance cannot occur. To define the mechanism by which H-NS interferes at this step in the initiation pathway, the architecture of the RNAP⅐H-NS⅐DNA complex was analyzed by scanning force microscopy (SFM). The SFM images show that the DNA flanking the RNA polymerase in open initiation complexes is bridged by H-NS. On the basis of these data, we present a model for the specific repression of transcription initation at the rrnB P1 by H-NS.

“…4, c and d). Thus, the mutant did not acquire the ability to repress the P2 promoter solely through the occupancy of O E (10) or though a roadblock mechanism in which transcription arrests because of GalR binding to O I . 1 The mutant GalR protein could form a loop with increased stability if protein-protein interactions between either two GalR dimers or GalR and HU were enhanced.…”

Section: Figmentioning

confidence: 93%

Genetic Analysis of GalR Tetramerization in DNA Looping during Repressosome Assembly

Geanacopoulos

2002

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The Gal repressosome is a nucleoprotein complex consisting of 2 GalR dimers, 1 HU, and 1 DNA loop, which represses the transcription of the gal operon. We have adopted a structure-based genetic approach to complement ongoing physical studies of the complex. Homology-based and subsequent alanine-scanning mutageneses suggest that five residues in the DNA-distal subdomain of GalR dimer are important for repressosome formation. A further analysis of these and intragenic suppressors of looping-defective GalR mutants as well as gain-of-function mutants that permit repressosome assembly in the absence of HU show that GalR dimers contact each other in the repressosome in a partially stacked configuration.The formation of DNA loops through the binding of regulatory proteins to spatially separated sites is a common feature in DNA transaction reactions (1-6). In both prokaryotes and eukaryotes, the components of many nucleoprotein complexes containing DNA loops have been characterized, but higher order structures are largely unknown. Questions such as the characteristic modes by which DNA loops form and the way in which the abundant architectural components of the nucleoid contribute to DNA loops remain to be resolved (3, 4).The Gal repressosome containing a DNA loop is one such complex whose function is to represses the transcription of the genes for galactose metabolism in Escherichia coli (7-9). Its assembly requires (i) the binding of two dimeric GalR repressors to two operators flanking the two gal promoters, (ii) the cooperative binding of HU, an abundant component of the nucleoid to a site between the two operators, and (iii) negatively supercoiled DNA. Fig. 1a shows the gal regulatory region. In our attempts to understand the structure of the Gal repressosome, we have adopted a genetic approach, which we hope will complement the structural studies and provide critical in vivo information on how the repressosome forms. In this report, we present the results of homology-based site-directed mutagenesis and alanine scanning of the GalR surface as well as allele-specific suppression genetics to characterize the domains of GalR dimers that interact to form a tetramer while bound to DNA. The results confirm that repressosome formation involves several residues on the DNA-distal edge of the GalR dimer that make critical contacts in the higher order complex. EXPERIMENTAL PROCEDURESBacterial Strains and Plasmids-The strains and plasmids used in this study have been previously described with one exception (8). The galP2ϳgusA reporter fusion, in which O E is deleted, was constructed by two rounds of PCR amplification. A primer, which completely replaced O E , and a primer complementary to the sequence upstream of the EcoR1 site in pI24 (8) were used to amplify the DNA upstream of the operator, and the complement to the mutagenic primer and a primer complementary to galE were used to amplify the downstream sequence including the HindIII site in galE. These PCR products were annealed and amplified with the primers complementary ...